Prion protein amino acid sequence influences formation of authentic synthetic PrPSc

Synthetic prions, generated de novo from minimal, non-infectious components, cause bona fide prion disease in animals. Transmission of synthetic prions to hosts expressing syngeneic PrPC results in extended, variable incubation periods and incomplete attack rates. In contrast, murine synthetic prions (MSP) generated via PMCA with minimal cofactors readily infected mice and hamsters and rapidly adapted to both species. To investigate if hamster synthetic prions (HSP) generated under the same conditions as the MSP are also highly infectious, we inoculated hamsters with HSP generated with either hamster wild type or mutant (ΔG54, ΔG54/M139I, M139I/I205M) recombinant PrP. None of the inoculated hamsters developed clinical signs of prion disease, however, brain homogenate from HSPWT- and HSPΔG54-infected hamsters contained PrPSc, indicating subclinical infection. Serial passage in hamsters resulted in clinical disease at second passage accompanied by changes in incubation period and PrPSc conformational stability between second and third passage. These data suggest the HSP, in contrast to the MSP, are not comprised of PrPSc, and instead generate authentic PrPSc via deformed templating. Differences in infectivity between the MSP and HSP suggest that, under similar generation conditions, the amino acid sequence of PrP influences generation of authentic PrPSc.

PrP conformation that, through an inefficient process of generating folding intermediates, results in atypical PK-resistant PrP (i.e., PrP res ) prior to production of authentic PrP Sc24, 25 . An exception to this is the murine synthetic prions (MSP) produced under PMCA conditions that are highly infectious for mice and can efficiently cross the species barrier to hamsters, suggesting these MSPs are bona fide PrP Sc15, 26 . Here, we investigated whether hamster synthetic prions, created using the same process as the highly infectious MSPs, were infectious for hamsters.

Emergence of clinical infection and slow adaptation of HSP to hamsters. CNS material from
hamsters subclinically infected with HSP WT or HSP ΔG54 was serially passaged twice in hamsters. Groups of hamsters (n = 5 per group) were i.c. inoculated with UN, HSP WT -, or HSP ΔG54 -infected brain (2nd passage) or spinal cord (3 rd passage) homogenate. All (n = 5) hamsters inoculated with either hamster passaged HSP WT (HaHSP WT ) or HSP ΔG54 (HaHSP ΔG54 ) at both serial passages developed clinical signs of prion infection with incubation periods of 335 ± 6 and 305 ± 5 dpi at second passage and 168 ± 3 dpi and 315 ± 10 dpi at third passage, respectively (Fig. 2, Table 1). Hamsters inoculated with UN brain homogenate remained clinically normal for more than 400 dpi for both serial passages. Clinical disease progression was prolonged, with a clinical phase of 41 ± 3 and 60 ± 2 days at second passage and 57 ± 3 and 81 ± 10 days at third passage for HaHSP WT -and HaHSP ΔG54 -infected hamsters, respectively (Table 1). At second passage, HaHSP WT -and HaHSP ΔG54 -infected hamsters were characterized clinically by ataxia, lethargy, and progressive weight gain (Table 1). However, one animal inoculated with HaHSP ΔG54 presented with hyperexcitability and lacked the progressive weight gain observed in the other HaHSP ΔG54 -infected animals. By the third serial hamster passage, clinical signs of HaHSP WT -infected hamsters included hyperexcitability and a trembling that developed into ataxia. In contrast, HaHSP ΔG54 -infected hamsters at third passage were clinically characterized by a mild hyperexcitability that developed into lethargy. Progressive weight gain remained a shared clinical characteristic of HaHSP WT -and HaHSP ΔG54 -infected hamsters. Overall, clinical prion disease was established at second passage for both HaHSP WT -and HaHSP ΔG54 -infected hamsters and HaHSP WT -and HaHSP ΔG54 -infected hamsters were clinically similar until third passage.  Survival curves depicting the changes in the incubation periods of HaHSP WT -and HaHSP ΔG54 -infected hamsters during adaptation. The incubation periods of both HaHSP WT -and HaHSP ΔG54 -infected hamsters at second passage were prolonged (335 ± 6 and 305 ± 5 dpi, respectively). However, at third passage, the incubation period of HaHSP WT -infected hamsters shortened to 168 ± 3 dpi. In contrast, the incubation period of HaHSP ΔG54infected hamsters at third passage remained relatively stable (315 ± 10 dpi). This divergence of incubation periods at third passage corresponds to divergence in clinical signs as well.   Fig. 4). At third passage, the average conformational stability [Gdn-HCl] ½ of PrP Sc from the CNS of HaHSP WT -and HaHSP ΔG54 -infected hamsters increased to 2.26 ± 0.01 and 2.14 ± 0.03 M, respectively, significantly (p < 0.05) more stable than PrP Sc from the CNS of DY-infected hamsters but significantly (p < 0.05) less stable than PrP Sc from the CNS of HY-infected hamsters (Fig. 4). The average conformational stability of PrP Sc from both HaHSP WT -and HaHSP ΔG54 -infected hamsters increased between second and third passage. This is in contrast to PrP Sc from hamsters infected with murine synthetic prions, which remained stable throughout serial passage 26 . Overall, the conformational stability of PrP Sc changed throughout serial passage and is intermediate between DY and HY controls.

HaHSP WT -and HaHSP ΔG54 -infected hamsters are characterized by the classical neuropathological hallmarks of prion disease. Hematoxylin and eosin staining of HaHSP WT -and
HaHSP ΔG54 -infected brain sections revealed characteristic spongiosis associated with prion disease (Fig. 5b, c) whereas brains from mock-infected animals lacked spongiosis (Fig. 5A). Immunohistochemistry (IHC) with the anti-PrP antibody 3F4 identified abnormal prion protein deposition in the brains of HaHSP WT -and HaHSP ΔG54 -infected animals (Fig. 5E, F) but was not identified in mock-infected animals (Fig. 5D). Compared to brain sections from mock-infected animals (Fig. 5G, J), HaHSP WT -and HaHSP ΔG54 -infected brain sections also showed astrogliosis (Fig. 5H, I) and microgliosis (Fig. 5K, L) when the astrocyte marker GFAP and microglia marker Iba-1 were utilized in IHC, respectively. Overall, HaHSP WT -and HaHSP ΔG54 -infected hamsters exhibit the neuropathological hallmarks of prion disease, similar to animals infected with brain-derived prions.

Discussion
Murine and hamster synthetic prions (MSP and HSP, respectively) produced under identical conditions have vastly different capacities for establishing prion disease and adapting to hamsters. Bacterially-generated murine or hamster recombinant PrP (recPrP) underwent serial PMCA in the presence of RNA and an endogenous lipid, POPG to produce MSP and HSP 15 . Interspecies transmission of the MSP to hamsters was more efficient than intraspecies transmission of other synthetic prions 14,17,18,20,24,26 . Additionally, the MSP rapidly adapted to hamsters and the biochemical characteristics of PrP Sc from hamster-adapted MSP (HaMSP) remained stable throughout serial passage. These results were consistent with the intraspecies transmission of the MSP to mice, which resulted in a 100% attack rate and relatively short, stable incubation period at first passage 15 . Overall, the rapid adaptation www.nature.com/scientificreports/ of the MSP to both mice and hamsters and the stability of clinical and biochemical characteristics suggested the MSP are high titer, authentic PrP Sc composed of a single strain. In stark contrast, in the current study, hamsters inoculated with HSP WT failed to develop clinical signs of prion disease at first passage, but subclinical infection was indicated by the presence of PK-resistant PrP in brains of HSP WT -infected hamsters (Fig. 1). HSP WT adapted slowly to hamsters. Clinical onset occurred at second passage following an extended (335 ± 6 dpi) incubation period, which shortened (168 ± 3 dpi) by third passage. In contrast to the MSP, the conformational stability of PrP Sc from HSP WT -infected hamsters increased as the incubation period decreased. Fluctuations in conformational stability during serial passage is observed with other synthetic prions as well 18,20,31 . Dynamic PrP Sc conformational stability during serial passage suggests continual adaptation of HSP WT to hamsters and selection of a dominant strain from a mixture. We hypothesize that the PMCA cofactors and conditions used to generate the MSP and HSP WT favored formation of authentic PrP Sc using murine recPrP but not hamster recPrP, with HSP WT triggering formation of bona fide PrP Sc through the process of deformed templating.
The deformed templating conversion model is consistent with the observed transmission properties of HSP to hamsters. Both SSLOW (Synthetic Strain Leading to OverWeight)-, the prototypic strain of the deformed templating model, and HSP WT -infected hamsters did not develop clinical signs of prion infection at first passage, instead developing clinical signs at second passage following an extended incubation period 18 . The conformational Figure 5. Brains of HaHSP WT -or HaHSP ΔG54 -infected hamsters are characterized by the histopathological hallmarks of prion disease. Brain sections from mock-infected (UN), second passage HaHSP WT -, and second passage HaHSP ΔG54 -infected animals were stained with hematoxylin and eosin (a-c) to observe spongiform degeneration. Immunohistochemistry was also performed using the anti-PrP antibody 3F4 (d-f), the astrocyte marker GFAP (g-i), and the microglial marker Iba-1 (j-l) to observe abnormal PrP deposition, astrogliosis, and microgliosis, respectively. The white schematic inset in (A) depicts the brain region imaged in (a-c). The black schematic in (d) depicts the brain region imaged in (d-l). Scale bar 100 μm; inset scale bar 25 μm. www.nature.com/scientificreports/ stability of PrP Sc from SSLOW-and HSP WT -infected hamsters changed throughout adaptation, decreasing or increasing, respectively, as the incubation period shortened. Although SSLOW and HSP share similarities, they have markedly different neuropathology. The neuropathology of SSLOW-infected hamsters is characterized by large PrP Sc deposits, which are notably absent in HSP neuropathology (Fig. 5) 18,32 . Transmission of HSP WT and SSLOW to hamsters is similar, suggesting HSP WT established prion disease via deformed templating. The serial seeded generation of the HSP could also be considered a deformed templating process. The MSP could not directly seed hamster WT recPrP, but instead could seed double mutant hamster recPrP. The double mutant synthetic prions then could seed single mutant hamster recPrP and the single mutant synthetic prions could seed hamster WT recPrP. We hypothesize this step-wise generation process of the HSP may produce synthetic prions at a different point in the deformed templating process (i.e., atypical PrP-res versus fibrillar amyloid). As SSLOW synthetic prions are hypothesized to be fibrillar amyloid, the HSP being further along in the deformed templating process could account for observed differences in establishment of infection and adaptation between the two synthetic prions. Additionally, differences between SSLOW and HSP could result from differences in generation conditions, with SSLOW synthetic prions formed de novo under denaturing and shaking conditions whereas HSP formed using PMCA. PMCA can expedite interspecies transmission and adaptation of brain-derived prions and thus could expedite the deformed templating process 33 . We cannot exclude the possibility that HSP contains low titer PrP Sc , however, we think this is unlikely as we would expect PMCA to generate higher titer material of a single strain similar to what we observed with MSP 26 . Overall, despite differences in generation of and disease phenotype caused by the HSP and SSLOW, both utilize a similar conversion pathway to establish infection. Structural differences between synthetic prions and brain-derived PrP Sc could explain the need for synthetic prions to utilize deformed templating to generate authentic PrP Sc . Recent cryo-EM studies have revealed the structures of both synthetic prion fibrils formed from human recombinant PrP and infectious PrP Sc derived from patient samples [34][35][36][37] . The monomeric structures of human synthetic prion fibrils and brain-derived PrP Sc from GSS patients differed, as did interfacing of the monomers within the protofilaments comprising the prion fibrils [34][35][36][37] . This could explain the observed differences in infectivity, in which the synthetic fibril structure is less efficient at PrP Sc conversion. This incongruity has also been observed in α-synuclein. Inoculation of brain homogenate from MSA patients to TgM83 +/− mice results in an average incubation of ~ 120 dpi whereas inoculation of preformed fibrils to TgM83 +/− mice results in highly variable incubation periods ranging from ~ 90 to 330 dpi [38][39][40] . The differences in incubation period correspond to differences in structure between the preformed fibrils and patient-derived α-synuclein [41][42][43] . Overall, the structural disparity between in vitro generated fibrils and brain-derived prions is a possible explanation for the inefficiency of synthetic prions following intraspecies transmission and supports the role of deformed templating in establishing infection.
Mutations in the hamster recPrP used to generate the HSP affected infectivity and strain emergence. Amino acid sequence differences between murine and hamster PrP at residues 54, 139, and 205 greatly impact the mouse/hamster species barrier. In the current study, the mutations introduced to hamster PrP (ΔG54, ΔG54/ M139I, and M139I/I205M) increased the similarity of the hamster PrP amino acid sequence to the murine PrP sequence. Of the three HSPs generated using mutant hamster recPrP, only the HSP ΔG54 mutant caused subclinical infection, whereas the other mutants, HSP ΔG54/M139I and HSP M139I/I205M , failed to establish infection in hamsters. Interestingly, HSP ΔG54/M139I and HSP M139I/I205M share a substitution of the hamster methionine at residue 139 for the murine isoleucine (Supplemental Fig. S1). Studies in Sc + -MNB cells, cell-free conversion systems, and transgenic mice have found that methionine or isoleucine expressed at residue 138/139 (murine/ hamster numbering) confers resistance or susceptibility to prion infection [44][45][46] . This susceptibility or resistance depends on which PrP background a mutation is introduced (mouse or hamster) and the strain of PrP Sc used to test conversion [44][45][46] . Recent cryo-EM data comparing the structure of anchorless RML mouse and 263 K hamster PrP Sc highlights the structural differences between these strains at residue 138/139 1,47 . The substitution of isoleucine at residue 139 in hamster recombinant PrP may affect the structure of the resulting HSP, subsequently affecting its ability to seed conversion of hamster WT PrP C . Taken together, these studies indicate residue 139 plays an important role in the mouse/hamster species barrier and mutation at this residue may hinder conversion of hamster PrP C to PrP Sc via deformed templating.

Ethics statement. All procedures involving animals were approved by the Creighton University
Institutional Animal Care and Use Committee and comply with the Guide for the Care and Use of Laboratory Animals and ARRIVE guidelines. Synthetic prions. The murine synthetic prions (MSP) were generated as previously described 15,19,26,48 .
Briefly, murine recombinant PrP (PrP23-230) was expressed in E. coli and purified 48 . Murine recombinant PrP (25 μg/ml in deionized H2O), 1-palmitoyl-2-oleoylphophatidylglycerol (POPG; 22.2 μg/ml in 20 mM Tris HCl, pH 7.4), and total RNA isolated from mouse liver (150 μg/ml) were mixed in buffer (deionized H 2 O, 5% Triton X-100, and 10 × TN buffer) prior to serial PMCA that consisted of 30 s of sonication followed by 29.5 min incubation (one round is 24 h) 15,19,49 . Four hamster synthetic prions (HSP) were generated using either hamster WT or mutated recombinant PrP. The mouse and hamster PrP amino acid sequences differ at 12 residues. Analysis of which of these residues has the greatest effect on the mouse/hamster species barrier, assessed by changes in PMCA conversion efficiency, determined residues 54, 139, and 205 (hamster numbering) have the greatest impact. The mutations to the hamster recombinant PrP amino acid sequence are as follows: (1) deletion of glycine at residue 54 (HSP ΔG54 ); (2) deletion of glycine at residue 54 and substitution of methionine with isoleucine at residue 139 (HSP ΔG54/M139I ); (3) substitution of methionine with isoleucine at residue 139 and a substitution of isoleucine with methionine at residue 205 (HSP M139I/I205M ) (Supplemental Fig. S1). These www.nature.com/scientificreports/ mutations to the hamster PrP amino acid sequence increased the similarity of the hamster sequence to the murine sequence. The hamster synthetic prions were generated in PMCA using the same buffer and cofactors (RNA and POPG) as the de novo generated MSP, but were serially converted by seeded conversion. The MSP seeded conversion of hamster double mutant recombinant PrP, double mutant HSP seeded conversion of hamster single mutant recombinant PrP, and single mutant HSP seeded conversion of hamster WT recombinant PrP.
Animal bioassay. Groups of male, 3-4 week Syrian hamsters (n = 5 per group) were inoculated with 25 μl of murine or hamster synthetic prions 15,19 or a 10% (wt/vol) brain or spinal cord homogenate by the intracranial (i.c.) inoculation route as previously described 50 . Spinal cord homogenate was used as inoculum for third passage as whole brains were collected for histology at second passage. Hamsters were monitored three times per week for onset of clinical signs of prion disease. Incubation period was calculated as the number of days between inoculation and onset of clinical signs of prion infection. Clinical duration of disease was calculated as the number of days between onset of clinical signs and sacrifice. Animals were weighed once per week. Hamsters were considered moribund when weight declined for more than three weeks in a row or hamsters lost greater than 10 g in 1 week.
Tissue collection and processing. Moribund animals were anesthetized with isoflurane (Patterson Veterinary, Loveland, CO) and perfused transcardially with Dulbecco's phosphate-buffered saline (DPBS; Corning, Manassas, VA). Following euthanasia, tissues were collected using strain-dedicated tools that were decontaminated by immersion in bleach (neat) for 15 min at room temperature. Brains were collected whole for histology, collecting spinal cord (C1-C3) for biochemistry.

SDS-PAGE and western blot.
Detection of PrP Sc by Western blot was performed as previously described 26 .
Briefly, 5% w/v brain homogenate was incubated with proteinase K (PK; 100 μg/mL stock; Roche Diagnostics, Mannheim, Germany) for 1 h at 37 °C with shaking. To halt PK digestion, an equal volume of 2 × sample buffer Conformational stability assay. The PrP Sc conformational stability assay was performed as described previously 26,52  Immunohistochemistry. Immunohistochemistry (IHC) was performed as previously described 26,28 .
Briefly, brain sections were deparaffinized and incubated in formic acid (Sigma-Aldrich, St. Louis, MO) for 10 min. To block endogenous peroxidases, slides were incubated in 0.3% v/v H 2 O 2 in methanol for 20 min at room temperature. To block non-specific binding, sections were incubated in 10% vol/vol normal horse (or goat) serum (Vector, Burlingame, CA) in TTBS for 30 min at room temperature. Sections were incubated with either the monoclonal anti-PrP antibody 3F4 51 (final concentration of 3.33 μg/mL; EMD Millipore, Billerica, MA), anti-glial fibrillary acidic protein antibody (GFAP; final concentration of 1.45 μg/mL; Abcam, Cambridge, MA), or anti-Iba1 antibody (final concentration of 0.67 μg/mL; DakoCytomation, Glostrup, Denmark) overnight at 4 °C. Sections were next incubated with either horse or goat anti-mouse biotinylated antibody (1:700; Vector, Burlingame, CA) for 30 min at room temperature followed by ABC solution (Vector, Burlingame, CA) for 20 min at room temperature. The chromogen was developed with 0.05% w/v DAB (3,3′-Diaminobenzidine) in tris-buffered saline (TBS) with 0.003% v/v or 0.0015% v/v H 2 O 2 in MilliQ water and counterstained with hematoxylin. Images of brain sections were captured as described above.

Equipment and settings. For Figs. 1 and 3a, Western blots were developed using Pierce SuperSignal West
Femto maximum-sensitivity substrate per manufacturer's instructions (Pierce, Rockford, IL) and imaged on a Li-Cor Odyssey Fc Imager (Li-Cor, Lincoln, NE) using the chemiluminescence channel. Exposure was increased to visualize low levels of PrP Sc and the unglycosylated bands for Figs. 1 and 3a, respectively. For Fig. 5, images of brain sections were captured using an Infinity 2 microscope camera (Teledyne Lumenera, Ottawa, ON) attached to a Nikon Eclipse 80i compound microscope (Nikon Instruments, Melville, NY) and ImageJ software. Main images were captured at 10 × magnification and inset images were captured at 40 × magnification. Dimensions of all images at capture were 2448 × 2048 pixels. Images were processed identically for white balance using Adobe Lightroom (Adobe, San Jose, CA).
Statistical analysis. Differences among groups for biochemical properties such as conformational stability was determined by one-way ANOVA (p < 0.05) using GraphPad Prism (GraphPad Software, San Diego, CA).

Data availability
All relevant data are within the manuscript and its Supporting Information files.